CN114831108A - Fresh tissue preservation solution and preparation method and application thereof - Google Patents
Fresh tissue preservation solution and preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
- Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation
Abstract
The invention discloses a fresh tissue preservation solution, which belongs to the technical field of biological sample preservation, and comprises an ionic buffer solution, tissue cell anabolic nutrients, carbohydrate compounds, an antioxidant and anti-apoptosis agent, wherein anions in the ionic buffer solution comprise acid radical ions and hydroxyl ions; the tissue cell anabolic nutrients include amino acids, nucleosides, and/or nucleotides; the saccharide compound is at least one of monosaccharide, disaccharide and polysaccharide; the antioxidant and anti-apoptosis agent comprises vitamins, vitamin derivatives, glutathione and/or allopurinol, and the pH value of the fresh combined preservation solution is 7.2-7.6. The fresh tissue preservation solution is used for preserving biological samples, so that the low-temperature preservation (2-8 ℃) and transportation of fresh tissues of human and animals can be effectively realized, and the preservation and transportation cost is greatly reduced.
Description
Technical Field
The invention belongs to the technical field of biological sample preservation, and particularly relates to a fresh tissue preservation solution as well as a preparation method and application thereof.
Background
Biological samples are the basis of life science research, and the quality of the biological samples largely determines the accuracy and reliability of research results. With the rapid progress of research means such as genomics, transcriptomics and proteomics, various analysis techniques also put higher demands on the quality of biological samples.
The quality of the biological sample is greatly influenced by the storage and transportation of the biological sample. At present, biological samples and extraction products thereof mainly have two main storage modes of ultralow temperature and normal temperature.
First and second ultra-low temperature preservation
Compared with a tissue fixed by formalin, the biomacromolecule with higher quality can be extracted from the tissue frozen and stored at low temperature so as to meet the requirement of the modern detection technology. However, studies have shown that a significant decrease in protein activity occurs in tissues after cryopreservation at-25 ℃ for about 1 year. In contrast, cryopreservation at cryogenic temperatures (-196 to-80 ℃) is more effective and has become a widely accepted method for long-term storage of biological samples. But the deep low temperature storage has high requirements on the freezing storage solution, the storage conditions are harsh, the storage and transportation cost is high, and the like, so that the application value and the scientific research and transformation capability of the sample are reduced.
Second, preservation at room temperature
The normal temperature preservation technology utilizes certain special technologies or containers to place the dehydrated and dried sample in a closed environment to isolate harmful factors such as water, air, microorganisms and the like; or adding a protective agent into the biological sample, wherein the protective agent permeates into the tissue cells to inactivate related enzymes, so that the purpose of protecting the sample in a normal temperature environment is achieved.
With the advent of the era of precision therapy, single Cell technology will play its role in many areas, such as large-scale single Cell sequencing projects like Human Cell Atlas (HCA) and Human cancer Atlas Network (HTAN), and applications in the fields of Tumor, immunity, microbiology, neuroscience, etc. Single-cell sequencing (Single-cell sequencing) refers to a sequencing technology for acquiring genetic information of a Single cell, namely, extraction, amplification and high-throughput sequencing analysis are performed on a genome or a transcriptome on a Single cell level. The technology can reveal the unique gene structure and gene expression state of a single cell, including data such as structural variation, copy number variation, RNA expression level and the like, so that different cell types can be accurately distinguished, and the technology is helpful for scientists to research molecular mechanisms at a cellular level.
The single cell sequencing has extremely high requirements on sample freshness, if cell suspension preparation cannot be immediately carried out after sampling, if transportation is required, or temporary storage and subsequent samples are required to be subjected to the same batch of experiments and the like, the samples need to be stored/transported under appropriate conditions, otherwise the most original tissue cell state of the samples cannot be ensured, and certain influence is caused on subsequent results.
Therefore, in single cell sequencing, factors in various aspects such as sample type, economy, space, time and the like are comprehensively considered, an optimal preservation mode is selected, sample quality is protected, and smooth completion of an experiment is further ensured.
Disclosure of Invention
In order to solve the above technical problems, the present invention aims to provide a novel preservation solution and preservation method for fresh tissue samples, so as to maintain the tissue samples with high cell activity and integrity of gene information, maintain the spatial structure of tissues, and simultaneously, not change the expression of tissue cell genes.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention provides a fresh tissue preservation solution in a first aspect, which comprises an ionic buffer solution, tissue cell anabolic nutrients, carbohydrate compounds and antioxidant and anti-apoptosis agents, wherein anions in the ionic buffer solution comprise acid radical ions and hydroxyl ions; the tissue cell anabolic nutrients include amino acids, nucleosides, and/or nucleotides; the saccharide compound is at least one of monosaccharide, disaccharide and polysaccharide; the antioxidant and anti-apoptosis agent comprises vitamins, vitamin derivatives, glutathione and/or allopurinol, and the pH value of the fresh combined preservation solution is 7.2-7.6.
In the present invention, the ionic buffer is used for maintaining the tissue cell osmotic pressure, buffering and regulating the pH value. In some embodiments of the invention, the cation in the ionic buffer comprises sodium and/or potassium ions. In some embodiments of the invention, the acid ion is selected from at least one of the group consisting of carbonate, bicarbonate, phosphate, hydrogen phosphate, dihydrogen phosphate, formate, acetate, permanganate, manganate, hydrochloride, chlorate, chlorite, hydrogen oxalate, oxalate. Thus, materials for preparing the ionic buffer include, but are not limited to, sodium carbonate, sodium bicarbonate, sodium phosphate, sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium formate, sodium acetate, sodium permanganate, sodium manganate, sodium chlorate, sodium chlorite, sodium oxalate, potassium carbonate, potassium bicarbonate, potassium phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, potassium formate, potassium acetate, potassium permanganate, potassium manganate, potassium hydrochloride, potassium chlorate, potassium chlorite, potassium hydrogen oxalate, and potassium oxalate.
In some embodiments of the invention, the amino acid is selected from at least one of glutamine, glycine, serine, tyrosine, asparagine, leucine.
In some embodiments of the invention, the nucleoside is selected from one of adenosine, guanosine, uridine, cytidine and thymidine.
In the present invention, the saccharide compound may be a monosaccharide, disaccharide or polysaccharide, or a mixture of the same kind of saccharide or different kinds of saccharide, such as a mixture of several monosaccharides, or a mixture of several disaccharides, or a mixture of several polysaccharides, or a mixture of one or more monosaccharides and one or more disaccharides, or a mixture of one or more monosaccharides and one or more polysaccharides, or a mixture of one or more disaccharides and one or more polysaccharides, or a mixture of one or more monosaccharides, one or more disaccharides and one or more polysaccharides. In some embodiments of the invention, the monosaccharide is selected from the group consisting of arabinose, ribose, xylose, lyxose, glucose, mannose, fructose, galactose, the disaccharide is selected from the group consisting of sucrose, trehalose, lactose, and the polysaccharide is dextran and/or starch.
In some embodiments of the invention, the vitamin is selected from at least one of vitamin a, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin or niacinamide), vitamin B6 (pyridoxine, pyridoxal or pyridoxamine), vitamin B7 (biotin), vitamin B9 (folic acid), vitamin B12 (cobalamin, hydroxycobalamin or methylcobalamin), vitamin C (ascorbic acid), vitamin D (cholecalciferol), vitamin E (tocopherol or triple bond tocopherol), and vitamin K (phylloquinone). In some preferred embodiments of the present invention, the vitamin is selected from at least one of riboflavin, niacinamide, vitamin C, vitamin D, water-soluble vitamin E folic acid.
In some preferred embodiments of the present invention, in the fresh tissue preservation solution, the cations in the ionic buffer solution include sodium ions and potassium ions, and the anions include hydrochloride ions, hydrogen phosphate ions, dihydrogen phosphate ions, and hydroxide ions; the tissue cell anabolic nutrients include glycine, leucine, and adenosine; the saccharide compounds are glucose, starch and trehalose; the antioxidant and anti-apoptotic agents include vitamin B9, vitamin C and glutathione.
In some more preferred embodiments of the present invention, the fresh tissue preservation solution comprises potassium chloride 0.1-0.4g/L, sodium hydrogen phosphate 0.1-0.4g/L, sodium dihydrogen phosphate 0.1-0.4g/L, potassium hydroxide 0.1-0.4g/L, glucose 1.0-4.0g/L, starch 10.0-40.0g/L, trehalose 10.0-40.0g/L, glycine 10-30mg/L, leucine 10-30mg/L, adenosine 10-30mg/L, glutathione 0.1-1.0mg/L, folic acid 0.1-1.0mg/L, vitamin C0.1-1.0 mg/L.
A second aspect of the present invention provides the method for preparing a fresh tissue preservation solution according to the first aspect of the present invention, comprising the steps of:
s1, adding the substances into a container;
s2, fixing the volume by using nuclease-free water;
s3, adjusting the pH value of the prepared solution to 7.2-7.6.
In a third aspect, the present invention provides the use of a fresh tissue preservation solution according to any one of the first aspect of the present invention for preserving fresh tissue.
In some embodiments of the invention, the fresh tissue is selected from at least one of the group comprising heart, brain, kidney, liver, lung, stomach, intestine, spleen, body tissue, tumor tissue.
The invention has the advantages of
Compared with the prior art, the invention has the following beneficial effects:
(1) the fresh tissue preservation solution disclosed by the invention is simple in components, easy to prepare, easy to obtain raw materials, low in price and long in service life.
(2) The fresh tissue preservation solution can maintain the activity of the tissue cells, does not change the gene expression and the cell morphology of the tissue cells, and the preserved tissue sample can be directly used for subsequent experiments without resuscitation.
(3) The fresh tissue preservation solution is used for preserving biological samples, and is different from the traditional ultra-low temperature cryopreservation (80 ℃ below zero or liquid nitrogen), so that the low-temperature preservation (2-8 ℃) and transportation of the fresh tissues of human and animals can be effectively realized, and the preservation and transportation cost is greatly reduced.
Drawings
FIG. 1 shows the results of RNA detection after preservation of tissues for 24 hours in fresh Tissue preservation solutions #1 to #3 and MACS Tissue Storage Solution in example 2 of the present invention, A: fresh tissue preservation fluid # 1; b: fresh tissue preservation fluid # 2; c: fresh tissue preservation fluid # 3; d: fresh tissue preservation fluid # 4; e: fresh tissue preservation fluid # 5; f: CN111418581A preservation solution; g: MACS Tissue Storage Solution.
FIG. 2 shows the results of RNA detection after 48 hours of preservation of fresh Tissue preservation solutions #1 to #3 and MACS Tissue Storage Solution in example 2 of the present invention, A: fresh tissue preservation fluid # 1; b: fresh tissue preservation fluid # 2; c: fresh tissue preservation fluid # 3; d: CN111418581A preservation solution; e: MACS Tissue Storage Solution.
FIG. 3 shows the results of RNA detection 72 hours after fresh Tissue preservation solutions #1 to #3 and MACS Tissue Storage Solution in example 2 of the present invention, A: fresh tissue preservation fluid # 1; b: fresh tissue preservation fluid # 2; c: fresh tissue preservation fluid # 3; d: MACS Tissue Storage Solution.
FIG. 4 shows the results of DNA detection 72 hours after preservation of tissues with fresh Tissue preservation solutions #1 to #3 and MACS Tissue Storage Solution in example 2 of the present invention, M: DNA marker; a: fresh tissue preservation fluid # 1; b: fresh tissue preservation fluid # 2; c: fresh tissue preservation fluid # 3; d: fresh tissue preservation fluid # 4; e: fresh tissue preservation fluid # 5; f: CN111418581A preservation solution; g: MACS Tissue Storage Solution.
FIG. 5 shows the results of RNA detection after Storage of the Tissue of the center, brain, kidney, stomach, intestine and spleen in fresh Tissue preservation Solution #1 and MACS Tissue Storage Solution for 24 hours in example 3 of the present invention.
FIG. 6 shows the results of RNA detection in the center, brain, kidney, stomach, intestine and spleen tissues after 48 hours preservation in fresh Tissue preservation Solution #1 and MACS Tissue Storage Solution in example 3 of the present invention.
FIG. 7 shows the results of the dissociated cell viability (AO/PI staining) assay of the liver, brain, heart and intestine tissues of the middle mouse in example 4 of the present invention after preservation in fresh tissue preservation solution # 1.
FIG. 8 shows the results of dissociated cell survival (AO/PI staining) assays of liver, brain, heart and intestine tissues of mice in example 4 of the present invention after Storage in a MACS Tissue Storage Solution.
FIG. 9 shows the AO/PI staining assay of dissociated cells of liver, brain, heart and intestine tissues of middle mouse in example 4 of the present invention after 72 hours of preservation in fresh Tissue preservation Solution #1 and MACS Tissue Storage Solution.
FIG. 10 shows the results of RNA-Seq of tissues 0 hour and 48 hours after the preservation of mouse spleen and liver tissues in fresh tissue preservation solution #1 in example 5 of the present invention.
FIG. 11 shows the results of RNA-Seq of tissues after 0 hour and 48 hours of preservation of spleen and liver tissues of mice in MACS Tissue Storage Solution in example 5 of the present invention.
FIG. 12 shows the result of OCT embedded section staining after mouse heart tissue was stored in fresh tissue storage solution #1 for 48 hours in example 6 of the present invention.
FIG. 13 shows the result of staining OCT embedded sections after the brain tissue of mice in example 6 of the present invention was stored in fresh tissue storage solution #1 for 48 hours.
FIG. 14 shows the results of the survival rate of dissociated single cells after preservation of human lung cancer, colorectal cancer and breast cancer in fresh tissue preservation solution #1 for 48 hours in example 7 of the present invention.
FIG. 15 shows the results of the dissociated single cell viability after 48h of preservation of human lung, colorectal and breast cancer in MACS Tissue Storage Solution in example 7 of the present invention.
FIG. 16 shows the AO/PI staining detection results of dissociated cells of human lung cancer, colorectal cancer and breast cancer in example 7 of the present invention after 48h of preservation in fresh tissue preservation solution # 1.
Detailed Description
Unless otherwise indicated, implied from the context, or customary in the art, all parts and percentages herein are by weight and the testing and characterization methods used are synchronized with the filing date of the present application. Where applicable, the contents of any patent, patent application, or publication referred to in this application are incorporated herein by reference in their entirety and their equivalent family patents are also incorporated by reference, especially as they disclose definitions relating to synthetic techniques, products and process designs, polymers, comonomers, initiators or catalysts, and the like, in the art. To the extent that a definition of a particular term disclosed in the prior art is inconsistent with any definitions provided herein, the definition of the term provided herein controls.
The numerical ranges in this application are approximations, and thus may include values outside of the ranges unless otherwise specified. A numerical range includes all numbers from the lower value to the upper value, in increments of 1 unit, provided that there is a separation of at least 2 units between any lower value and any higher value. For example, if a compositional, physical, or other property (e.g., molecular weight, melt index, etc.) is recited as 100 to 1000, it is intended that all individual values, e.g., 100, 101, 102, etc., and all subranges, e.g., 100 to 166, 155 to 170, 198 to 200, etc., are explicitly recited. For ranges containing a numerical value less than 1 or containing a fraction greater than 1 (e.g., 1.1, 1.5, etc.), then 1 unit is considered appropriate to be 0.0001, 0.001, 0.01, or 0.1. For ranges containing single digit numbers less than 10 (e.g., 1 to 5), 1 unit is typically considered 0.1. These are merely specific examples of what is intended to be expressed and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application.
When used with respect to chemical compounds, the singular includes all isomeric forms and vice versa (e.g., "hexane" includes all isomers of hexane, individually or collectively) unless expressly specified otherwise. In addition, unless explicitly stated otherwise, the use of the terms "a", "an" or "the" are intended to include the plural forms as well.
The terms "comprising," "including," "having," and derivatives thereof do not exclude the presence of any other component, step or procedure, and are not intended to exclude the presence of other elements, steps or procedures not expressly disclosed herein. To the extent that any doubt is eliminated, all compositions herein containing, including, or having the term "comprise" may contain any additional additive, adjuvant, or compound, unless expressly stated otherwise. Rather, the term "consisting essentially of … …" excludes any other components, steps or processes from the scope of any of the terms hereinafter recited, insofar as such terms are necessary for performance. The term "consisting of … …" does not include any components, steps or processes not specifically described or listed. Unless explicitly stated otherwise, the term "or" refers to the listed individual members or any combination thereof.
In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments.
Examples
The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the disclosures and references cited herein and the materials to which they refer are incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
The molecular biological experiments, which are not specifically described in the following examples, were performed according to the specific methods listed in the manual of molecular cloning, laboratory manual (fourth edition) (j. sambrook, m.r. green, 2017), or according to the kit and product instructions. Other experimental methods, unless otherwise specified, are conventional. The instruments used in the following examples are, unless otherwise specified, laboratory-standard instruments; the test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
EXAMPLE 1 preparation of different fresh tissue preservation solutions
Preparation of fresh tissue preservation solutions according to Table 1
TABLE 1 preparation of fresh tissue preservation solutions #1- #5
Further, the tissue preservation solution is prepared according to the formula disclosed in the Chinese patent application CN 111418581A. The formulation is shown in table 2:
TABLE 2 CN111418581A preservation solution compounding table
| Serial number | Component name | Concentration of |
| 1 | Potassium hydroxide | 5-6g |
| 2 | Sodium dihydrogen phosphate | 3-5g |
| 3 | Anhydrous magnesium sulfate | 0.5-1g |
| 4 | Lactobionic acid | 30-40g |
| 5 | Cotton seed candy | 15-20g |
| 6 | Hydroxyethyl starch | 50-60g |
| 7 | Adenosine (I) | 1-2g |
| 8 | Allopurinol | 0.2-0.5g |
| 9 | Glutathione | 0.5-2g |
| 11 | Nuclease-free water | Make up to 1000mL |
| 12 | Sodium hydroxide, hydrochloric acid | Adjusting the pH to 7.4 |
Mixing the above solutions, and storing at 2-8 deg.C.
Example 2 assessment of nucleic acid degradation after storage of mouse liver tissue
Fresh liver Tissue of a C57BL mouse was cut into Tissue pieces with a volume of not more than 1cm × 1cm and a weight of 0.25 g. + -. 10%, which were stored in 2mL of fresh Tissue preservation solutions #1 to #5, CN111418581A and MACS Tissue Storage Solution prepared in example 1, respectively, stored in a refrigerator at 2 to 8 ℃ and subjected to DNA and RNA extraction from Tissue samples taken at 24 hours, 48 hours and 72 hours, respectively. The Agilent2100 bioanalyzer performed RNA detection and tissue DNA was detected by agarose gel electrophoresis.
The results show that, when mouse liver tissues are stored in fresh Tissue Storage solutions #1 to #3 for 24 hours, the Tissue RNA is not significantly degraded, the results are equivalent to the CN111418581A Storage solutions and MACS Tissue Storage Solution in Storage effect, while the Tissue RNA is significantly degraded after the fresh Tissue Storage solutions #4 to #5 are stored for 24 hours (as shown in figure 1), the mouse liver tissues are stored in the fresh Tissue Storage solutions #1 to #3 for 48 hours, the Tissue RNA is not significantly degraded and is relatively complete, the results are equivalent to the MACS Tissue Storage Solution in Storage effect, and the liver Tissue RNA is significantly degraded after the mouse liver tissues are stored in the CN111418581A Storage Solution for 48 hours (as shown in figure 2), so that the subsequent Storage tests are not performed. After the tissues were stored in fresh Tissue preservation solutions #1 to #3 and MACS Tissue Storage Solution for 72 hours, the RNA degradation in fresh Tissue preservation Solution #1 was not significant, while the Tissue RNA degradation was significant in fresh Tissue preservation Solution #2, fresh Tissue preservation Solution #3 and MACS Tissue Storage Solution (as shown in FIG. 3). The agarose gel electrophoresis results all show that the DNA of the mouse liver tissue is not degraded after 7 kinds of preservation solutions are preserved for 72 hours (as shown in FIG. 4).
From the above quality inspection results, it is found that the effect of the fresh Tissue preservation Solution #1 is superior to that of the fresh Tissue preservation solutions #2 to #5, the CN111418581A preservation Solution and the MACS Tissue Storage Solution, and therefore the fresh Tissue preservation Solution #1 was selected as the test preservation Solution in the following examples, and the MACS Tissue Storage Solution was used as the control.
Example 3 RNA integrity test after storage of other visceral organs in mice
BALB/c mouse heart, brain, kidney, stomach, intestine and spleen tissues are respectively taken, processed into tissue blocks with the volume not more than 1cm multiplied by 1cm and the weight deviation not more than 10 percent, and respectively stored in 2mL of fresh tissue preservation solution #1 and MACS tissue storage solution at the temperature of 2-8 ℃. Samples were taken at 24 hours and 48 hours respectively for RNA extraction from tissue samples and tested for RNA integrity by an Agilent2100 bioanalyzer.
The results showed that no significant degradation of tissue RNA occurred after 24 hours, 48 hours storage of heart, brain, kidney, stomach, intestine, spleen tissue in fresh tissue storage solution #1, which is comparable to the macstissus setagensolution storage results (as shown in fig. 5 and 6).
Example 4 detection of cell Activity after tissue preservation in mice
BALB/c mouse liver, brain, heart and intestine tissues are respectively processed into Tissue blocks with the volume not more than 1cm multiplied by 1cm and the weight deviation not more than 10 percent, and the Tissue blocks are respectively stored in 2mL of fresh Tissue preservation Solution #1 and MACS Tissue Storage Solution and are stored at the temperature of 2-8 ℃. Tissues were taken at 24 hours, 48 hours, and 72 hours, respectively, for single cell dissociation, and AO/PI staining was performed to examine cell viability using a fluorescent cell counter.
As shown in FIGS. 7 and 8, the activity of liver, brain, heart and intestine tissues of mice preserved by the fresh tissue preservation solution #1 and the MACS tissue storage solution is higher than 90% within 24 hours, and very high cell activity is maintained; within 48 hours, the activity of the liver, brain, heart and intestine tissues of the mice preserved by the fresh tissue preservation solution #1 and the MACS tissue storage solution is higher than 80%, so that higher cell activity is maintained, and the sequencing requirement of a single-cell transcriptome is met. However, the preservation effect of the fresh tissue preservation solution #1 was significantly better than that of MACS tissue storage solution (FIGS. 7, 8 and 9) after 72 hours of preservation of each type of tissue.
Example 5 RNA-Seq detection of Gene expression in mice after preservation of fresh tissue
BALB/c mouse liver and intestine tissues are respectively processed into Tissue blocks with the volume not more than 1cm multiplied by 1cm and the weight deviation not more than 10 percent, and the Tissue blocks are respectively stored in 2mL of fresh Tissue preservation Solution #1 and MACS Tissue Storage Solution and are stored at the temperature of 2-8 ℃. And respectively taking tissues at 0 hour and 48 hours for RNA extraction, and detecting whether the gene expression is obviously different from that of a fresh tissue in the tissue storage process by using RNA-Seq.
As shown in fig. 10 and 11, the gene expression of the liver and intestine tissues of the mice preserved with the fresh tissue preservation solution #1 was not significantly changed after 48 hours of preservation, and was consistent with that of the original fresh samples. Meanwhile, compared with the MACS tissue storage solution, the tissue preserved by the fresh tissue preservation solution #1 can better keep the consistency of the gene expression of tissue cells, and can be used for carrying out subsequent single cell sequencing and space transcriptome sequencing experiments.
Example 6 histological cell morphology after mouse tissue preservation
BALB/c mouse brain and heart tissues are respectively processed into tissue blocks with the volume not more than 1cm multiplied by 1cm and the weight deviation not more than 10 percent, and the tissue blocks are respectively stored in 2mL of fresh tissue storage fluid #1 and stored at the temperature of 2-8 ℃. And taking the tissue at 48 hours, carrying out OCT embedding, sectioning and HE staining on the tissue, and carrying out section scanning on the tissue by using a digital pathological section scanner to observe the tissue cell morphology.
The results are shown in FIGS. 12 and 13, and the brain and heart tissues were preserved for 48 hours, with intact tissue morphology and no pathological damage.
Example 7 human clinical tissue preservation
Samples of human fresh clinical colorectal surgery, breast cancer surgery and lung cancer surgery are processed into tissue blocks with the volume not more than 1cm multiplied by 1cm and the weight deviation not more than 10 percent, and the tissue blocks are stored in 2ml of fresh tissue preservation solution #1 and MACS tissue storage solution and stored at the temperature of 2-8 ℃. The tissue was taken at 48 hours for single cell dissociation and AO/PI staining, and cell viability was measured by a fluorescent cell counter.
The results are shown in fig. 14, fig. 15 and fig. 16, after human colorectal, breast cancer and lung cancer tissues are preserved for 48 hours by the fresh tissue preservation solution #1, the survival rates of the tissue cells are all higher than 80%, which indicates that the fresh tissue preservation solution #1 can well maintain the tissue cell activity within 48 hours when human clinical tissue samples are preserved by the fresh tissue preservation solution # 1.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (10)
1. A fresh tissue preservation solution, characterized in that the fresh tissue preservation solution comprises an ionic buffer solution, a tissue cell anabolic nutrient, a carbohydrate, an antioxidant and an anti-apoptotic agent, wherein,
the anions in the ionic buffer solution comprise acid radical ions and hydroxide ions;
the tissue cell anabolic nutrients include amino acids, nucleosides, and/or nucleotides;
the saccharide compound is at least one of monosaccharide, disaccharide and polysaccharide;
the antioxidant and anti-apoptosis agent comprises vitamins, vitamin derivatives, glutathione and/or allopurinol,
the pH value of the fresh combined preservation solution is 7.2-7.6.
2. The fresh tissue preservation solution according to claim 1, wherein the acid ion is at least one selected from the group consisting of carbonate, bicarbonate, phosphate, hydrogen phosphate, dihydrogen phosphate, formate, acetate, permanganate, manganate, hydrochloride, chlorate, chlorite, oxalate, and oxalate.
3. The fresh tissue preservation solution according to claim 1, wherein the amino acid is at least one selected from the group consisting of glutamine, glycine, serine, tyrosine, asparagine, and leucine.
4. The fresh tissue preservation solution according to claim 1, wherein the nucleoside is one selected from adenosine, guanosine, uridine, cytidine and thymidine.
5. The fresh tissue preservation solution according to claim 1, wherein the monosaccharide is selected from the group consisting of arabinose, ribose, xylose, lyxose, glucose, mannose, fructose, galactose, the disaccharide is selected from the group consisting of sucrose, trehalose, lactose, and the polysaccharide is dextran and/or starch.
6. The fresh tissue preservation solution according to claim 1, wherein the vitamin is at least one selected from the group consisting of vitamin a, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D, vitamin E, and vitamin K.
7. The fresh tissue preservation solution according to claim 1, wherein in the fresh tissue preservation solution,
the positive ions in the ion buffer solution comprise sodium ions and potassium ions, the negative ions comprise hydrochloric acid radical ions, hydrogen phosphate radical ions, dihydrogen phosphate radical ions and hydroxyl ions, and the pH value of the ion buffer solution is 7.2-7.6;
the tissue cell anabolic nutrients include glycine, leucine, and adenosine;
the saccharide compounds are glucose, soluble starch and trehalose;
the antioxidant and anti-apoptotic agents include vitamin B9, vitamin C and glutathione.
8. The method for preparing a fresh tissue preservation solution according to claim 1, comprising the steps of:
s1, adding the substances into a container;
s2, fixing the volume by using nuclease-free water;
s3, adjusting the pH value of the prepared solution to 7.2-7.6.
9. Use of the fresh tissue preservation solution of claim 1 to preserve fresh tissue.
10. The use according to claim 9, wherein the fresh tissue is selected from at least one of the group comprising heart, brain, kidney, liver, lung, stomach, intestine, spleen, body tissue, tumor tissue.
Priority Applications (1)
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| CN107926933A (en) * | 2018-01-14 | 2018-04-20 | 上海赛立维生物科技有限公司 | A kind of cell-preservation liquid |
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